We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. Chemokine receptors signal predominantly by triggering G alpha i nucleotide exchange. Humans and mice have three Gi isoforms although Gi alphai2 (encoded by Gnai2) and Gi alphai3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2 -/- T and B cells have severe defects in chemokine-receptor signaling while Gnai2 +/- T and B cells exhibit modest defects. In vivo, the Gnai2-/- B cells fail to properly access lymph node follicles and the Gnai2-/- T cells failing to properly enter the T cell zone. We have crossed the Gnai2 targeted mice with the Rgs1 targeted mice. Analysis of the double heterozygote, Rgs1 +/- Gnai2 +/- indicates that the ratio between Gnai2 expression and Rgs1 expression regulates responsive of B lymphocytes to chemokines. In contrast to the Gnia2-/- B cells, Gnai3-/- B cells exhibit enhanced chemokine receptor signaling when compared with wild type B cells. At an organismal level, variations in Rgs1 and Gnai2 expression affect marginal zone B cell development, splenic architecture, lymphoid follicle size, and germinal center morphology. Gnai2 was also needed for the proper alignment of MOMA-1+ macrophages and MAdCAM-1+ endothelial cells along marginal zone sinuses in the spleen. To investigate the molecular basis of chemotactic properties of mammalian cells, we are examining the distribution of heterotrimeric G proteins and RGS proteins in living cells during chemotaxis. A confocal microscope-based cell migration assay has been established to visualize localization of fluorescent protein-tagged G protein signaling components in differentiated HL-60 cells. Treatment of HL-60 cells with 1.3% DMSO induces differentiation of these cells to neutrophil-like cells and caused changes in gene expression patterns of G protein signaling components. Treating the differentiated HL-60 cells with the chemoattractant fMetLeuPhe further altered the mRNA expression pattern of the RGS proteins. We are currently examining intracellular distribution of YFP tagged Gi alpha2 and G beta/gamma complexes, and GFP-tagged RGS1, RGS13, and RGS14 in addition to GRK2-GFP during dHL-60 cell migration. In addition constructs expressing Gi alpha2 proteins insensitive to RGS proteins or to GDI proteins have been made. The consequence of these mutations with respect to the ability of cells to migrate as well as possible changes in intracellular distribution of signaling components will be examined. We have also shown that BM-derived mouse neutrophils express many RGS proteins including RGS1, RGS10, RGS13, RGS14, and RGS19. Initial assessment indicates that Rgs13-/- neutrophils have enhanced responses to chemoattractants. Neutrophils from Rgs1 deficient and from Gi alpha2-G184S knock-in mice are also being assessed. Rgs1 has been linked to both type 1 diabetes and celiac disease. A prominent site of Rgs1 expression is in intraepithelial lymphocytes (IELs), which are found in the gastrointestinal tract (GI). IEL dysfunction has also been implicated in inflammatory diseases of the GI tract. Initial analysis of wild type and Rgs1-/- IELs revealed a modest increase in the numbers of IELs in the KO mice. Analysis of chemotaxis responses indicated little difference between wild type and KO IELs. The most striking difference is a marked increase in gamma-interferon production by cultured KO IELs versus similarly cultured wild type IELs. Currently immunohistochemical and microarray approaches are being used to analyze the differences between wild type and Rgs1 deficient IELs. These studies are being performed in collaboration with Dr. Adrian Hayday (King's College London Medical School). Germinal center B lymphocytes strongly express the RGS protein, RGS13. To facilitate our studies of RGS13 and to provide a potential resource for in vivo imaging endogenous germinal center B cells we have generated mice where GFP (green fluorescent protein) has been knocked-in (KI) into the Rgs13 locus replacing the RGS13 coding sequence. Flow cytometry revealed high levels of GFP expression in B220+ Fas+ GL7+ cells from the spleen, lymph nodes, and Peyers patches. Flow cytometry also revealed a marked increase in the number of B-T cell conjugates observed following immunization of the KI versus wild type mice. The Immunohistochemistry of GFP expression revealed RGS13 gene expression in the dark zone of germinal centers with reduced levels in the light zone region. A microarray analysis of RGS13 KI GFP positive B cells confirmed the high expression of GC specific genes and along with markedly upregulated cell cycle genes. The mice had elevated serum IgM, IgG2A, and IgG3 and increased number and size of Peyers patches. In the spleen germinal center morphology was altered with disorganized dark and light zones although overall size and number of germinal centers was similar to wild type. Although the levels of specific antibodies induced was not significantly disturbed the Rgs13-/- generated a higher affinity response than did wild type mice. The GFP+Rgs13-/- germinal center B cells have an enhanced response to chemoattractants compared to wild type cells. Intravital microscopy revealed GFP positive cells at sites consistent with germinal centers as well as a strongly GFP positive cells that appeared distinct from lymphocytes, many of which appeared along the edge of the B cell follicle. Labeled transferred wild type B cells were often observed to interact with these cells. The strongly GFP positive cells may be mast cells, which are known to express Rgs13. Analysis of bone marrow cells cultured under conditions known to generate mast cell revealed that approximately 15% of the cells expressed GFP and 98% expressed mast cell markers. RGS10 is strongly expressed in lymphocytes with the highest levels in thymocytes, CD4 T cells, and marginal zone B cells. Analysis of these mice has revealed surprisingly few differences when compared to littermate controls. Current studies are aimed understanding whether RGS10 has any role in inflammation as number of chemokine and interleukin genes are dysregulated in RGS10-deficient microglia from the central nervous system. Both Rgs10 deficient macrophages and T cells will be stimulated and subject to microarray to identify whether a similar dysregulation occurs. Rgs19 is well expressed in B lymphocytes and less so in T cells. It is also well expressed in stem cells and hematopoietic progenitors. GFP has been inserted into the RGS19 locus and appropriated targeted mice identified. Rgs19 GFP KI mice are born with a normal Mendelian frequency. Flow cytometry revealed that GFP is well expressed in mature B and T cells and in progenitors in the bone marrow. Analysis of B cell development in the bone marrow revealed a reduction in pre-B cells with normal numbers of early progenitors. In the spleen there is a modest increase in B220 positive cells and a substantial increase in T1 transitional B cells. Consistently the Rgs19 GFP KI mice had an enlarged spleen and thymus compared to wild type mice. Chemotaxis experiments comparing wild type and RGS19-/- lymphocytes indicate significant increases to a panel of chemokines with the KO B cells and a marginal increase with KO T cells. Current studies are aimed understanding the pathogenesis of the splenomegaly and enhanced B cell hematopoiesis in these mice.